Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B (NANB) viral hepatitis, infecting approximately 4 million people in the U.S. HCV has a positive-stranded RNA genome consisting of a single open reading frame (ORF) flanked by the untranslated regions (UTR) at the 5' and 3' ends. The ORF encodes a large polypeptide that is processed to structural and nonstructural viral proteins. The roles of viral proteins and conserved RNA sequences/structures in HCV replication are poorly understood. The overall goal of this research proposal is to determine the sequence and structural requirements of cis-acting RNA elements in the 5' and 3' UTRs in viral RNA replication. Both the 5' and 3' UTR sequences are highly conserved among various HCV isolates. Genetic studies have demonstrated that both 5' and 3' UTRs are essential for HCV RNA replication. We hypothesize that the conserved 5' and 3' UTRs contain cis-acting RNA elements important for control of HCV RNA replication. The highly conserved 5'UTR harbors two distinct RNA elements, a short 5'-proximal RNA element with a stern-loop structure and a longer element of internal ribosomal entry site (IRES). To determine cis-acting RNA elements in the 5'UTR and to define their roles in HCV RNA replication, we will perform systematic mutagenesis analyses of the conserved sequences/structures by nucleotide deletions, substitutions, and replacement with non-HCV IRES. The 3'UTR consists of three distinct regions, a highly conserved 98 nucleotides that form three stern-loop structures, a poly(U/C) tract of variable length, and a variable region at immediate downstream of the ORF. Cis-acting RNA elements in the 3'UTR for HCV RNA replication will be defined by nucleotide deletions and substitutions. The effects of mutations in the 5' and 3' UTRs on HCV RNA replication will be determined by using reverse genetics systems. We have developed a DNA-based HCV replicon replication system. We have also demonstrated that a DNA-based HCV 'minigenome' RNA was replicated when active replicase complex was provided in trans. These reverse genetics systems for HCV RNA replication will be used to determine cis-acting RNA elements in the 5' and 3' UTRs and their roles in HCV RNA replication. Information gained from these studies will immediately contribute to our understanding of the molecular mechanism of HCV RNA replication and provide meaningful insights to the design of effective strategies for controlling HCV infection.